1. Field of the Invention
This invention pertains to novel antibodies and antibody fragments that specifically bind to human Proprotein Convertase Subtilisin-like/Kexin type 9 (hereinafter “PCSK9”) and compositions containing. In addition the invention relates to nucleic acids encoding said antibodies and antibody fragments and the use thereof to express said antibodies and antibody fragments in desired host cells. Also, the invention relates to therapeutic and diagnostic use of these antibodies and antibody fragments.
More particularly, the invention provides rabbit antibodies and humanized and chimeric antibodies derived therefrom specific to PCSK9 as well as antibody fragments specific to PCSK9 which include e.g., Fab′, F(ab′)2, Fv, scFv fragments, SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies, monovalent antibodies such as MetMab like antibodies, and IgNAR.
Further, the invention provides nucleic acids and host cells containing that encode for and result in the expression of the subject anti-PCSK9 antibodies, i.e., rabbit antibodies and antibody fragments and modified forms thereof including by way of example humanized and chimeric antibodies derived therefrom as well as antibody fragments which include e.g., Fab′, F(ab′)2, Fv, scFv fragments, SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies, monovalent antibodies such as MetMab like antibodies, and IgNAR.
Also the invention relates to expression systems for the manufacture of the inventive anti-PCSK9 antibodies, including yeast, fungi, mammalian, and other cells useful for the manufacture of antibodies and antibody fragments.
Also, the invention relates to novel antibodies and antibody fragments that specifically bind to human PCSK9 which compete with and/or specifically bind to the same or overlapping epitope(s) on PCSK9 as any of the anti-PCSK9 antibodies and antibody fragments exemplified herein.
The invention further pertains to the in vivo use of the subject anti-PCSK9 antibodies and antibody fragments alone or in association with other active agents or drugs. for blocking, inhibiting or neutralizing PCSK9.
The invention further pertains to the in vivo use of the subject anti-PCSK9 antibodies and antibody fragments alone or in association with other active agents or drugs. for blocking or inhibiting the interaction of PCSK9 with LDLR.
The invention also specifically relates to methods for treating or preventing disorders of cholesterol or lipid homeostasis and disorders associated therewith including by way of example hypercholesterolemia, hyperlipidemia, hypertriglyceridaemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, metabolic syndrome, acute coronary syndrome, xanthoma, hypertension, angina, obesity, diabetes and vascular inflammation, by the administration of the subject anti-PCSK9 antibodies and antibody fragments, wherein the subject antibodies and antibody fragments may be used alone or in association with other active agents.
The invention further specifically relates to methods of preventing or treating diseases and disorders associated with PCSK9, e.g., diseases associated with increased or decreased levels of PCSK9 and/or mutations in the PCSK9 gene that affect PCSK9 protein expression, primary sequence and/or function by administering said antibodies or fragments thereof alone or in combination with other active agents.
The present invention further provides methods for improving blood cholesterol markers associated with increased risk of heart disease using the subject antibodies and antibody fragments alone or in association with other active agents. These markers include, but are not limited to, high total cholesterol, high LDL, high total cholesterol to HDL ratio and high LDL-C to HDL ratio.
The present invention further provides methods for treating or preventing any of the following conditions or complications associated therewith such as hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular diseases, Alzheimer's disease and generally dyslipidemias, which can be manifested, for example, by an elevated total serum cholesterol, elevated LDL, elevated triglycerides, elevated VLDL, and/or low HDL. Some non-limiting examples of primary and secondary dyslipidemias that can be treated using an antibody or antibody fragment according to the invention, either alone, or in combination with one or more other agents include the metabolic syndrome, diabetes mellitus, familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemias, including heterozygous hypercholesterolemia, homozygous hypercholesterolemia, familial defective apoplipoprotein B-100; polygenic hypercholesterolemia; remnant removal disease, hepatic lipase deficiency; dyslipidemia secondary to any of the following: dietary indiscretion, hypothyroidism, drugs including estrogen and progestin therapy, beta-blockers, and thiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia.
In addition, antibody or antibody fragment according to the invention can also be useful in preventing or treating atherosclerotic diseases, such as, for example, coronary heart disease, coronary artery disease, peripheral arterial disease, stroke (ischaemic and hemorrhagic), angina pectoris, or cerebrovascular disease and acute coronary syndrome, myocardial infarction. In some embodiments, the antibody or antibody fragment according to the invention is useful in reducing the risk of: nonfatal heart attacks, fatal and non-fatal strokes, certain types of heart surgery, hospitalization for heart failure, chest pain in patients with heart disease, and/or cardiovascular events because of established heart disease such as prior heart attack, prior heart surgery, and/or chest pain with evidence of clogged arteries. In some embodiments, the antibody or antibody fragment according to the invention and methods can be used to reduce the risk of recurrent cardiovascular events.
The invention also particularly relates to the use of the subject anti-PCSK9 antibodies and antibody fragments in any of the aforementioned therapeutic indications or conditions in combination with other drugs that are typically used to treat such disorders, wherein the antibody and other drug or agent may be co-administered or separately administered. Non limiting examples of drugs that may be co-administered with the subject antibodies or antibody fragments or used in the same therapeutic regimen include by way of example statins, ACE inhibitors, Angiotensin II receptor blockers (ARBs), Antiarrhythmics, Antiplatelet Drugs, aspirin, beta blockers, amiodarone, digoxin, aspirin, anti-clotting agents, digoxin, diuretics, heart failure drugs, vasodilators, blood thinners, other anti-cholesterol drugs such as holestyramine (Questran), gemfibrozil (Lopid, Gemcor), Omacor, and pantethine, other anti-hypertensives, antidiabetigenic drugs such as Alpha-glucosidase inhibitors, Biguanides, Dipeptidyl peptidase-4 inhibitors, Insulin therapies, Meglitinides, Sulfonylurea, and Thiazolidinediones, and other drugs used to treat conditions wherein the treated individual may have high cholesterol.
The invention further relates to compositions containing the subject anti-PCSK9 antibodies or antibody fragments, especially compositions are suitable for in vivo administration, e.g., subcutaneous, intravenous, intradermal, intranasal, rectal, vaginal, intrathecal, oral, and other administrable dosage forms.
The invention further relates to compositions containing the subject anti-PCSK9 antibodies or antibody fragments, especially compositions suitable for in vivo administration, e.g., subcutaneous, intravenous, intradermal, intranasal, rectal, vaginal, intrathecal, oral, and other administrable dosage forms which contain another active agent such as statins, ACE inhibitors, Angiotensin II receptor blockers (ARBs), Antiarrhythmics, Antiplatelet Drugs, aspirin, beta blockers, amiodarone, digoxin, aspirin, anti-clotting agents, digoxin, diuretics, heart failure drugs, vasodilators, blood thinners, other anti-cholesterol drugs such as holestyramine (Questran), gemfibrozil (Lopid, Gemcor), Omacor, and pantethine, other anti-hypertensives, antidiabetigenic drugs such as Alpha-glucosidase inhibitors, Biguanides, Dipeptidyl peptidase-4 inhibitors, Insulin therapies, Meglitinides, Sulfonylurea, and Thiazolidinediones, and other drugs used to treat conditions wherein the treated individual may have high or aberrant lipid or cholesterol levels.
The invention further relates to storage stable forms containing the subject anti-PCSK9 antibodies or antibody fragments, e.g., lyophilisates, suspensions, and buffered or temperature stable compositions.
The invention also pertains to methods of using the subject antibodies and antibody fragments that specifically bind to PCSK9 in screening assays to detect and monitor the levels of PCSK9 in serum samples, potentially for the diagnosis of diseases and disorders associated with PCSK9 or identifying individuals wherein the administration of the subject antibodies and antibody fragments that specifically bind to PCSK9 may have a therapeutic or prophylactic effect or for monitoring the effects of a treatment designed to modulate or neutralize PCSK9 or block cholesterol synthesis, e.g., a treatment involving administration of one of the subject anti-PCSK9 antibodies or antibody fragments.
2. Description of Related Art
Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serine protease involved in regulating the levels of the low density lipoprotein receptor (LDLR) protein (Horton et al., 32(2) Trends Biochem. Sci. 71-77 (2007); Seidah and Prat, 85(7) J. Mol. Med. 685-96 2007). In vitro experiments have shown that adding PCSK9 to HepG2 cells lowers the levels of cell surface LDLR (Benjannet et al., 279 J. Bio. Chem. 48865-75 (2004); Lagace et al., 116(11) J. Clin. Invest. 2995-3005 (2006); Maxwell et al., 102(6) Proc. Nat. Acad. Sci. 2069-74 (2005); Park et al., 279 J. Biol. Chem. 50630-38 (2004)). (While this protein is generally referred to as PCSK9, it is noted that this protein and corresponding gene has also been referred to in the patent and non-patent literature by other names including PSEC0052, FH3, HCHOLA3, LDLCQ1, NARC-1, NARC1, PC9).
Experiments with mice have shown that increasing PCSK9 protein levels decreases levels of LDLR protein in the liver (Benjannet et al., 279 J. Bio. Chem. 48865-75 (2004); Lagace et al., 116(11) J. Clin. Invest. 2995-3005 (2006); Maxwell et al., 102(6) Proc. Nat'l Acad. Sci. 2069-74 (2005); Park et al., 279 J. Biol. Chem. 50630-38 (2004)), while PCSK9 knockout mice have increased levels of LDLR in the liver (Rashid et al., 102(15) Proc. Nat'l Acad. Sci. 5374-79 (2005)). Additionally, various human PCSK9 mutations that result in either increased or decreased levels of plasma LDL-C have been identified (Kotowski et al., 78(3) Am. J. Hum. Genet. 410-22 (2006); Zhao et al., 79(3) Am. J. Hum. Genet. 514-23 (2006)). PCSK9 has been shown to directly interact with the LDLR protein, be endocytosed along with the LDLR, and co-immunofluoresce with the LDLR throughout the endosomal pathway (Lagace et al., 116(11) J. Clin. Invest. 2995-3005 (2006)). Several mutations in human PCSK9 cause gain-of-function effects in humans, including hypercholesterolemia, increased LDL-C cholesterol levels, and increased risk of coronary heart disease. (Gamier, 11(3) Am. J. Cardiovasc. Drugs 145-52 (2011)). Even rarer human PCSK9 mutations induce loss-of-function, resulting in lowered LDL-C levels and a 88% reduction in coronary heart disease risk. (Id.) PCSK9 interacts with the LDLR via the EGF domain and the complex is internalized. In the endosome, the lower pH results in an increased affinity between PCSK9 and LDLR and the complex is targeted for degradation in the lysosome (Horton et al., April Supp., J. Lipid Res. S172-177 (2009), Sci. 928-33 (2003)).
Several lines of evidence demonstrate that PCSK9, in particular, lowers the amount of hepatic LDLR protein and thus compromises the liver's ability to remove LDL cholesterol from the circulation. Adenovirus-mediated overexpression of PCSK9 in the livers of mice results in the accumulation of circulating LDL-C due to a dramatic loss of hepatic LDLR protein, with no effect on LDLR mRNA levels; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; Maxwell & Breslow, 2004 PNAS 101:7100-7105; Park et al., 2004 J. Biol. Chem. 279:50630-50638; and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319. The effect of PCSK9 overexpression on raising circulating LDL-C levels in mice is completely dependent on the expression of LDLR, again, indicating that the regulation of LDL-C by PCSK9 is mediated through downregulation of LDLR protein. In agreement with these findings, mice lacking PCSK9 or in which PCSK9 mRNA has been lowered by antisense oligonucleotide inhibitors have higher levels of hepatic LDLR protein and a greater ability to clear circulating LDL-C; Rashid et al., 2005 PNAS 102:5374-5379; and Graham et al., 2007 J. Lipid Res. 48(4):763-767. In addition, lowering PCSK9 levels in cultured human hepatocytes by siRNA also results in higher LDLR protein levels and an increased ability to take up LDL-C; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; and Lalanne et al., 2005 J Lipid Res. 46:1312-1319. Together, these data indicate that PCSK9 action leads to increased LDL-C by lowering LDLR protein levels.
A number of mutations in the gene PCSK9 have also been conclusively associated with autosomal dominant hypercholesterolemia (“ADH”), an inherited metabolism disorder characterized by marked elevations of low density lipoprotein (“LDL”) particles in the plasma which can lead to premature cardiovascular failure; see Abifadel et al., 2003 Nature Genetics 34:154-156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin. Genet. 65:419-422. A later-published study on the S127R mutation of Abifadel et al., supra, reported that patients carrying such a mutation exhibited higher total cholesterol and apoB100 in the plasma attributed to (1) an overproduction of apoB100-containing lipoproteins, such as low density lipoprotein (“LDL”), very low density lipoprotein (“VLDL”) and intermediate density lipoprotein (“IDL”), and (2) an associated reduction in clearance or conversion of said lipoproteins; Ouguerram et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1448-1453.
PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 100(3) Proc. Nat'l Acad. Sci. 928-33 (2003)). Humans have nine prohormone-proprotein convertases that can be divided between the S8A and S8B subfamilies (Rawlings et al., 34 Nucleic Acids Research D270-72 (2006)). Furin, PC1/PC3, PC2, PACE4, PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B. Crystal and NMR structures of different domains from mouse furin and PC1 reveal subtilisin-like pro- and catalytic domains, and a P domain directly C-terminal to the catalytic domain (Henrich et al., 10(7) Nature Structural Bio. 520-26 (2005); Tangrea et al., 320(4) J. Mol. Bio. 801-12 (2002)). Based on the amino acid sequence similarity within this subfamily, all seven members are predicted to have similar structures (Henrich et al., 345(2) J. Mol. Bio. 211-27 (2005)). SKI-1/S1P and PCSK9 are classified in subfamily S8A. Sequence comparisons with these proteins also suggest the presence of subtilisin-like pro- and catalytic domains (Sakai et al., 2(4) Mol. Cell 505-15 (1998); Seidah et al., 100(3) Proc. Nat'l Acad. Sci. 928-33 (2003); Seidah et al., 96(4) Proc. Nat'l Acad. Sci. 1321-26 (1999)). In these proteins, the amino acid sequence C-terminal to the catalytic domain is more variable and does not suggest the presence of a P domain.
Prohormone-proprotein convertases are expressed as zymogens, and they mature through a multi step process. The function of the pro-domain in this process is two-fold. The pro-domain first acts as a chaperone and is required for proper folding of the catalytic domain (Ikemura et al., 262 J. Biol. Chem. 7859-64 (1987)). Once the catalytic domain is folded, autocatalysis occurs between the pro-domain and catalytic domain. Following this initial cleavage reaction, the pro-domain remains bound to the catalytic domain where it then acts as an inhibitor of catalytic activity (Fu et al., 275 J. Biol. Chem. 16871-78 (2000)). When conditions are correct, maturation proceeds with a second autocatalytic event at a site within the pro-domain (Anderson et al., 16 Nature 1508-18 (1997)). After this second cleavage event occurs the pro-domain and catalytic domain dissociate, giving rise to an active protease.
Autocatalysis of the PCSK9 zymogen occurs between Gln152 and Ser153 (VFAQ|SIP) (Naureckiene et al., 420(1) Archives of Biochem. & Biophysics 55-67 (2003)), and has been shown to be required for its secretion from cells (Seidah et al., 100(3) Proc. Nat'l Acad. Sci. 928-33 (2003)). A second autocatalytic event at a site within PCSK9's pro-domain has not been observed. Purified PCSK9 is made up of two species that can be separated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolated without its inhibitory pro-domain, and measurements of PCSK9's catalytic activity have been variable (Naureckiene et al., 420(1) Archives of Biochem. & Biophysics 55-67 (2003); Seidah et al., 100(3) Proc. Nat'l Acad. Sci. 928-33 (2003)).
Accordingly, there is substantial evidence indicating that PCSK9 plays a role in the regulation of LDL; that the expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, that the corresponding inhibition or lack of expression of PCSK9 is associated with reduced LDL cholesterol plasma levels; and that decreased levels of LDL cholesterol are associated with sequence variations in PCSK9 have been found to confer protection against coronary heart disease; Cohen, 2006 N. Engl. J. Med. 354:1264-1272.
In clinical trials, reductions in LDL cholesterol levels have been directly related to the rate of coronary events; Law et al., 2003 BMJ 326:1423-1427. Also, moderate lifelong reduction in plasma LDL cholesterol levels was found to correlate with a substantial reduction in the incidence of coronary events; Cohen et al., supra. This was the case even in populations with a high prevalence of non-lipid-related cardiovascular risk factors; supra. Accordingly, there is great benefit to be reaped from the managed control of LDL cholesterol levels.
Based thereon, the identification of other molecules which may be used to modulate cholesterol levels and block or inhibit or neutralize the activity of PCSK9 would be of great interest. The present invention advances these interests by providing novel antagonists of PCSK9 for use for in blocking, inhibiting or neutralizing one or more of the activities of PCSK9 and/or in blocking the interaction of PCSK9 with LDLR and/or for the treatment of therapeutic conditions identified herein especially those involving or associated with high or aberrant lipid or cholesterol levels.